In vertebrates, movement requires that spinal motor neurons form precise connections with target muscles. In both embryonic development and nerve regeneration, axons find their targets by integrating signals from guidance cues to navigate a highly structured extracellular environment. These cues are generated by cells neighboring the prospective axonal path and are bound by or built into the extracellular matrix (ECM) to guide axons and establish neuromuscular synapses. Although much research has focused on delineating 'classical'axon guidance pathways mediated by cues like Netrins, Ephrins, and Slits, the mechanisms by which ECM components and their modifications direct axons during embryonic outgrowth and regeneration are not fully understood. The goal of this proposal is to define the molecular and cellular processes governed by ECM components and their modifications to guide spinal motor axons during development and regeneration. In zebrafish, recent studies demonstrate that mutations in lh3/diwanka, a glycosyltransferase that is expressed in cells bordering the axonal path, lead to axon pathfinding errors. Moreover, loss-of-function experiments suggest that lh3/diwanka provides guidance through the ECM proteoglycan Collagen XVIII (col18a1). Col18a1 is an atypical collagen that contains protein interaction domains for several signaling molecules important for axon pathfinding. Finally, ECM proteins, including col18a1, are upregulated at axon injury sites and along the path taken by regenerating axons, consistent with a possible function during axonal regeneration. In this proposal, I will define the mechanisms that ECM components and their modifications utilize to direct spinal motor axons during development and regeneration. First I will determine the temporal requirements of ECM modifications in guiding two distinct classes of spinal motor axons. Secondly, I will determine the col18a1 signaling mechanism(s) in motor axon guidance by defining the protein interaction domains that are necessary for proper motor axon guidance. Lastly, I will determine the role of lh3/diwanka- mediated ECM modifications in nerve regeneration by comparing the rate and fidelity of wild type nerve regrowth to that in the absence of these modifications. Together these experiments will define ECM mediated guidance pathways necessary for the development and reconstitution of neuromuscular connections. PUBLIC HEALTH RELEVANCE: Victims of spinal cord injury suffer from lifelong loss of movement and sensation because damaged nerves in the spinal cord fail to regenerate. In contrast, sheared nerves peripheral to the spinal cord regenerate and reinitiate sensory and motor activity. Results from these studies will define mechanisms that guide peripheral nerves to their targets with the hope that these pathways will provide avenues to new therapeutic treatment for patients suffering from nerve damage, including spinal cord injury.